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Es Cell International Pte Ltd

Academisch Ziekenhuis Leiden

Ncardia B.v.

Christine Lindsay Mummery

Robert Passier

Robert Zweigerdt

Bruce Paul Davidson

Xiuqin Xu

Ralph Eberhard Graichen

William Sun

Stefan Robbert Braam

Leon Tertoolen

Valeriya Viktorovna Orlova

This disclosure generally concerns the fields of cell biology and molecular biology. In particular the invention concerns the field of stem cell biology and maturation of stem cell-derived cardiomyocytes. Disclosed is a method for improving the maturity of stem-cell derived cardiomyocytes, in particular of the ventricular type, as can be witnessed by, for example, an improved upstroke velocity of the stem-cell derived cardiomyocytes.

Cardiomyocyte maturation

The disclosure is concerned among others with means and methods for obtaining endothelial cells and to means and methods for in vitro cell culture comprising endothelial cells and pericytes and/or smooth muscle cells derived from the pericytes. The endothelial cells or the pericytes and/or smooth muscle cells, or both, are preferably derived from in vitro differentiated pluripotent stem cells.

Differentiation and expansion of endothelial cells from pluripotent stem cells and the in vitro formation of vasculature like structures

This invention provides a method for testing a factor for cardiogenicity which comprises differentiating human embryonic stem (hES) cells to cardiomyocytes in the presence and absence of the factor wherein the human embryonic stem (hES) cells are cultured under a serum free condition comprising co-culture in the presence of END-2 cells or serum-free extracellular medium therefrom, and measuring the differentiation in the presence and absence of the factor. This invention also provides a method for identifying a cardiogenic factor, which comprises differentiating human embryonic stem (hES) cells to cardiomyocytes in the presence or absence of the factor wherein the human embryonic stem (hES) cells are cultured under a serum free condition comprising co-culture in the presence of END-2 cells or serum-free extracellular medium therefrom, and identifying the factor that affects the differentiation of human embryonic stem (hES) cells to cardiomyocytes in the presence or absence of the factor.

Cardiomyocyte differentiation

The present invention relates to the induction of differentiation in human embryonic stem (hES) cells to cardiomyocytes and factors such as prostaglandin I2 (PGI2), Fgf 9, Bmp6, Bmp4, Scf, Igf2, and insulin that influence the process of differentiation to cardiomyocytes. Media that is appropriate for the induction of differentiation of cardiomyocytes from hES cells is also provided wherein the media contains these factors. Genes that are upregulated in the process of cardiomyocyte differentiation are also provided.

Direct differentiation of cardiomyocytes from embryonic stem cells

The present invention relates to methods of inducing differentiation of stem cells. In particular, the invention relates to methods of inducing differentiation of embryonic stem cells into muscle cells or vascular endothelial cells. The invention also includes cells, cell lines, testing models and culture systems used in the methods of the present invention and differentiated cells produced therefrom. The present invention also provides methods of using the differentiated cells of the present invention for therapeutic purposes.

Methods of inducing differentiation of stem cells

The present invention relates to the induction of differentiation in stem cells to cardiomyocytes and factors such as prostaglandin alone or in combination with other factors including essential minerals selected from the group including transferrin and selenium, small molecules selected from the group including a p38 MAPK inhibitor such as SB203580 and protein growth factors of the FGF, IGF and BMP families such as but not limited to IGF1, FGF2, BMP2, BMP4 and BMP6. and insulin that influence the process of differentiation to cardiomyocytes. Media that is appropriate for the induction of differentiation of cardiomyocytes from stem cells is also provided wherein the media contains these factors. The use of cardiomyocytes and cardiac progenitors produced by the directed differentiation in transplantation and screening for cardiac compounds is also provided.

Prostacyclin directed differentiation of cardiomyocytes from human embryonic stem cells

The present invention provides a method of enhancing the efficiency of differentiation of hES cells into cardiomyocytes which method comprises incubating the cells under serum free conditions. The method typically includes providing cells that induce cardiomyocyte differentiation by cell to cell contact. Differentiation to cardiomyocytes can occur via two routes, namely by spontaneous differentiation and by induced differentiation. Without wishing to be bound by theory the present inventors hypothesize that, in the case of induced differentiation, END-2 cells, for instance, are needed for aggregation to cause local high cell densities and in inducing differentiation of nascent mesoderm. This second step could be enhanced in any human embryonic stem cell line leading to the prediction that it will work in lines other than hES. In cell lines that undergo spontaneous differentiation, it is hypothesized that local induction of embryoid bodies in endoderm occurs. Typically for induced differentiation this method will also comprise culturing the hES cell with a cell excreting at least one cardiomyocyte differentiation inducing factor or with an extracellular medium therefrom, under conditions that induce differentiation.

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DJ Bilthoven, Netherlands

Christine Lindsay Mummery